Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Investigating the structure and function of transmembrane domains in MARCH E3 ligases and single‑span receptors (#241)

Raphael Trenker 1 2 , Matthew E Call 1 2 , Melissa J Call 1 2
  1. Walter and Eliza Hall Institute of medical research, Parkville, VIC, Australia
  2. Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia

The mechanisms of assembly and function for many important type I/II (single-pass) transmembrane (TM) receptors are proposed to involve the formation and/or alteration of specific interfaces among their membrane-embedded alpha-helical TM domains. What these intramembrane interfaces look like and how they gain specificity is only poorly understood because of substantial technical challenges regarding their structural and functional characterisation. Isolated TM fragments are generally considered poor candidates for crystallisation due to their high hydrophobicity, flexible non-helical flanking sequences, low abundance of potential crystal contacts and the requirement for high lipid or detergent content in samples for screening. However, our lab has recently determined structures of TM complexes derived from glycophorin A1 and the immunoreceptor signalling adaptor DAP122 in monoolein lipidic cubic phase (LCP), demonstrating that TM complexes can be crystallised in a lipid bilayer and that native TM helix-helix interfaces are faithfully recapitulated under these conditions3. Since this provides compelling support for the wider application of LCP media for structural analysis of small TM complexes, we are currently using the technique in combination with LCP-FRAP (Fluorescence Recovery After Photobleaching) analysis to obtain structural information about other cell-surface receptors and related regulatory proteins that act via TM helix‑helix interactions. To complement structural studies, we further established Deep Mutational Scanning analysis of TM segments as a high‑throughput method to obtain mutational landscapes that characterise such interactions in great detail.

  1. Trenker R, Call ME, Call MJ. Crystal Structure of the Glycophorin A Transmembrane Dimer in Lipidic Cubic Phase. J Am Chem Soc. 2015 Dec 23;137(50):15676-9.
  2. Knoblich K, Park S, Lutfi M, van 't Hag L, Conn CE, Seabrook SA, Newman J, Czabotar PE, Im W, Call ME, Call MJ. Transmembrane Complexes of DAP12 Crystallized in Lipid Membranes Provide Insights into Control of Oligomerization in Immunoreceptor Assembly. Cell Rep. 2015 May 26;11(8):1184-92
  3. Trenker R, Call MJ, Call ME. Progress and prospects for structural studies of transmembrane interactions in single-spanning receptors. Curr Opin Struct Biol. 2016 Aug;39:115-123